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WO2007015690A1 - Séquençage de protéine/peptide par dégradation chimique en phase gazeuse - Google Patents

Séquençage de protéine/peptide par dégradation chimique en phase gazeuse Download PDF

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WO2007015690A1
WO2007015690A1 PCT/US2005/025185 US2005025185W WO2007015690A1 WO 2007015690 A1 WO2007015690 A1 WO 2007015690A1 US 2005025185 W US2005025185 W US 2005025185W WO 2007015690 A1 WO2007015690 A1 WO 2007015690A1
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ion
polypeptide
protein
mass
reactant
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Xiaoyu Chen
Michael S. Westphall
Lloyd M. Smith
Brian L. Frey
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Wisconsin Alumni Research Foundation
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Wisconsin Alumni Research Foundation
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6818—Sequencing of polypeptides
    • G01N33/6824—Sequencing of polypeptides involving N-terminal degradation, e.g. Edman degradation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry

Definitions

  • the invention relates to a method and corresponding apparatus for sequencing polypeptides and proteins in the gas phase using Edman degradation chemistry and mass spectrometry.
  • Proteins are among the most important components of all living systems. Some proteins are hormones; some help defend the body against damage or attack; others act as structural materials of cell walls and membranes, bone and cartilage, hoof and claw.
  • the building blocks of proteins consist of twenty amino acids, linked together by peptide bonds in chains. The diversity in form and function of proteins and peptides
  • the twenty naturally-occurring amino acids include side chains that are acidic (Asp and GIu), basic (Lys, Arg, and His), neutral/non-polar (GIy, Ala, VaI, Leu, He, Phe, Pro, Met), and neutral/polar (Ser, Thr, Tyr, Trp, Asn, GIn, and Cys).
  • the functional nature of a protein is determined by the folded structure that the amino acid polymer assumes.
  • the final three- dimensional form of a protein is largely dependent on its primary structure, i.e. the sequence of the various amino acids along the length of the protein molecule.
  • a phenylisothiocyanate is coupled to the ⁇ -amine of the protein or polypeptide to be sequenced.
  • the resulting phenylthiocarbamoyl (PTC) derivative is then hydrolyzed to yield the anilinothiazolinone (ATZ) derivative.
  • the ATZ derivative is then converted in aqueous acid to the more stable phenylthiohydantoin (PTH) (see Fig. 1).
  • PTH phenylthiohydantoin
  • the purified protein or polypeptide is applied to glass fiber disks and loaded directly into the reaction chamber cartridge of a gas-liquid solid phase sequencer. 4 If the sample is impure, as is commonly the case, gel electrophoresis is usually used to separate the mixture components. Purified protein sample is then transblotted onto chemically inert membranes, which are then placed in the sequencer for analysis. 5
  • reagents and solvents are delivered to the reaction cell under the control of a microprocessor.
  • Polar reagents are introduced in the gas phase to reduce sample loss.
  • the N-terminal ATZ derivative is extracted from the reaction cell and delivered into a conversion flask where it is converted to the more stable PTH amino acid. This final product is subsequently analyzed by HPLC. The elution time of the PTH-amino acid derivative is compared with that of standards to identify each residue.
  • Mass spectrometry is an analytical technique that determines the mass of atoms or molecules by means of ion-field (electric or magnetic) interactions.
  • a mass spectrometer consists of three fundamental components: An ionization source, where gas-phase ions are generated; a mass analyzer, where ions of different mass-to-charge ratios (m/z) are separated; and a detector, where the separated ions produce detectable signals.
  • MALDI The success of MALDI is based on the use of a matrix compound that absorbs laser irradiation at a wavelength where the analytes do not.
  • the analyte is co-crystallized with a small organic compound.
  • a laser pulse with sufficient energy density, a sudden and explosive phase transition occurs.
  • From among all the analyte molecules desorbed from the matrix only a small portion ( " 1O 4 ) are ionized.
  • 10 ' n gas-phase proton transfer is generally believed to be involved in this process. Ions produced in MALDI are usually singly-charged, making MALDI amenable to mixture analysis.
  • Electrospray ionization results in a distribution of multiply-charged ions for each analyte present.
  • the basic ESI source consists of a metal needle maintained at high voltage ( ⁇ 4 kV). The needle is positioned in front of a counter-electrode held at ground or low potential (and which also doubles as the inlet of the mass spectrometer). Sample solution is gently pumped through the needle and is transformed into a mist of micrometer-sized droplets that fly rapidly toward the counter electrode (see Fig. 2). In addition to the applied voltage, a concentric flow of nitrogen is often used to help nebulize the solution and dissolve the analyte ions. As each droplet decreases in size, the field density on its surface increases. When charge repulsion exceeds the force of surface tension, the parent droplet splits into smaller daughter droplets. This droplet fission continues until naked ions are formed.
  • Mass Analyzers MALDI and ESI have been coupled to many different mass analyzer types. The two most common are the Time Of Flight (TOF) and the Triple Quadrupole (QqQ).
  • TOF Time Of Flight
  • QqQ Triple Quadrupole
  • Time-of-flight is the simplest mass analyzer, consisting only of a metal flight tube.
  • the mass-to-charge ratios (m/z) of ions are determined by measuring the time it takes the ions to travel from source to detector.
  • m/z mass-to-charge ratios
  • TOF MS include the capability to deliver complete mass spectra at high speed and with no mass range limit.
  • the mass-resolving power in TOF measurement is, however, limited by the distribution of initial energy in the analyte molecules and the position of the ions prior to acceleration.
  • the spatial focusing plane in a single-stage mass spectrometer is only a short distance from the acceleration region (i.e., the apparatus has a relatively short focal length), after which the ions will spread out.
  • a two-stage acceleration system is often utilized to allow spatial focusing at a longer distances from the ion source.
  • the spatial focusing plane can be brought to the detector plane by adjustment of the relative field strength between these acceleration stages.
  • energy focusing can be achieved by the technique of delayed extraction, also known as time-lag focusing.
  • the most successful energy focusing method implemented to date is the "reflectron.”
  • an electrostatic ion mirror (the reflectron) is disposed at the distal end of the flight tube and the electrostatic field within the reflectron is oriented to oppose the acceleration field.
  • the accelerated ions penetrate into the reflectron, and are ultimately reflected back toward a secondary (or "reflected") focal point.
  • the more energetic ions penetrate more deeply into the reflectron and hence take longer to be reflected back out of the reflectrcn.
  • the optics can be adjusted to bring ions of different energies to a space-time focus. While the addition of a mirror provides little improvement in theoretical resolution, it dramatically broadens the mass range of focus. 12"14
  • a triple quadrupole mass spectrometer is comprised of two mass analyzing quadrupoles (Ql and Q3) and a radiofrequency-only quadrupole, q2 (see Fig. 3).
  • Quadrupole mass filters can be operated in two basic modes: mass-resolving mode and radio frequency only (RF-only) mode.
  • mass-resolving mode quadrupoles are operated at a constant ratio.
  • the operation points lie on a straight line in a stability diagram, known as the mass scan line (see Fig. 4).
  • the mass scan line can be viewed as a collection of points representing particles with different mass-to- charge ratios: heavier ions at the left-lower region and lighter ions at the right-upper region.
  • the portion of the mass scan line that is intercepted by the boundary of the stable region represents a transmission window. Only m/z ratios that fall into this window will be transmitted. The length of this segment defines the resolution of transmission.
  • the RF-only quadrupole functions as a collision cell in which the buffer gas pressure is maintained at about from 1 to about 119 mTorr.
  • Precursor ions selected by Ql enter the RF collision quadrupole, q2, where they undergo collision-induced dissociation.
  • Product ions are then mass filtered by scanning the third quadrupole, Q3, to produce the product mass spectrum.
  • Ion Detectors The most commonly used ion detectors are electron multiplier detectors, including channel electron multipliers (CEM) and microchannel plate detectors (MCP). These detectors operate by means of secondary electron generation. Initial secondary electrons generated upon impact of incident ions start an electron avalanche that produces an output signal. Because the response of electron multiplier detectors to ions with a fixed kinetic energy falls off significantly with increasing mass, ion detectors based on different detection mechanisms have been developed. One strategy is to detect the charge directly. Briefly, as ions approach the detector, image charges are formed on the surface of the detector, which are then picked up by an external circuit generating an output signal. The major limitation in this detection scheme is the low sensitivity due to the lack of inherent amplification.
  • the energy deposited in a suitable material by impact of an ion can be detected.
  • 16"24 Using two superconducting layers separated by an insulating layer, ions that strike the detector create non-thermal phonons (lattice vibrations). Phonons with sufficiently high energy can break the weakly bound electron pairs (Cooper pairs) in the superconducting layer, which results in a measurable tunneling current through the insulating baffler.
  • These detectors are more efficient than MCP's, especially for detecting large ions.
  • these types of detectors require liquid helium cooling and generally have a small active area, which limits their use in routine applications.
  • MS mass spectrometric
  • MS-based methods present a wholly new approach to protein and polypeptide sequencing.
  • Mass spectrometric (MS) approaches to protein sequencing fall into three general categories.
  • the first approach is to replace fluorescence detection with MS detection for gel electrophoresis. This approach is comparatively straightforward, but does not eliminate gel electrophoresis from the sequencing process.
  • the second approach is to replace gel electrophoresis with laser fluorescence, a more robust detection method.
  • the third approach (and the focus of the present invention) is to introduce an intact polypeptide or protein molecule into a mass spectrometer, to fragment the molecule, and then to determine the primary amino acid sequence of the molecule via mass spectral analysis of the fragments. This method is an enormous advancement over prior art amino acid sequencing methods.
  • mass spectrometry is simply introduced as a detection system. HPLC fractions of Edman degradation products are analyzed by a mass spectrometer in place of the conventional UV detector. Although the sensitivity has been pushed down to the high attomole level by using this method, the sequencing speed is not increased at all.
  • a concentrated set of peptide fragments (a sequencing ladder) is generated, either chemically or enzymatically, in a controlled fashion.
  • the sequencing ladder is subsequently separated and detected by a mass spectrometer.
  • This protein ladder sequencing technique lends itself to very high sample throughput at very low per-cycle cost. Disadvantages of this technique include the lengthy sample preparation and the large amount of pure peptide required.
  • Protein/peptide sequence information can also be obtained by tandem mass spectrometry. 26 ' 27
  • protein is first digested into peptide fragments with an enzyme. This mixture is subsequently introduced into the ion source of a mass spectrometer, either directly or after separation by liquid chromatography.
  • Precursor ions selected by a first mass analyzer, are fragmented by collision-induced dissociation (CID) or post-source decay (PSD). The resultant fragments are then analyzed by a second mass analyzer.
  • CID collision-induced dissociation
  • PSD post-source decay
  • MS acts as a powerful tool for identification of known proteins.
  • the protein of interest is first digested by a proteolytic enzyme and analyzed by mass spectrometric techniques.
  • the mass spectra of intact peptides constitute a "peptide mass fingerprint" (PMF) unique to the protein digested.
  • PMF peptide mass fingerprint
  • the PMF obtained is subsequently compared to "virtual" fingerprints derived by theoretical cleavage of protein sequences stored in a database.
  • the protein of interest is identified when a match is found.
  • the peptide mixture from protein digestion (usually by trypsin) is fractionated by either gel electrophoresis or liquid chromatography methods, the fractions of which are then analyzed by tandem MS.
  • the information created by the CID of peptides is used to search a protein database for a match within the expected MS/MS data from the known tryptic peptides.
  • the field of mass spectrometry has developed significant bioanalytical capacity with the recent development of the twin ionization techniques: MALDI and ESI.
  • MALDI and ESI twin ionization techniques
  • the mass spectrometric approaches based on database searching have become the method of choice in high-throughput identification of known proteins. These methods, however, will not work if the protein in question is not in a protein database.
  • the present invention is directed to a novel protein/polypeptide sequencing technique based on gas-phase Edman degradation.. This technique disclosed herein provides matchless speed and sensitivity for de novo sequence analysis of intact proteins and peptides.
  • sequence analysis of proteins is done using the Edman degradation to generate N-terminal PTH derivatives, whose identity is then determine using any number of methods (including mass spectrometry).
  • MS is used solely as a means for detection.
  • the Edman degradation reaction does not take place within the mass spectrometer itself.
  • Mass spectrometry can be coupled to Edman degradation in several different ways.
  • One approach is to use MS to replace the conventional UV detection at the end of each Edman degradation cycle. 29 As noted above, while this approach provides sensitivity at high attomolar level, there is no gain in sequencing speed.
  • Another approach is to generate sequencing ladders consisting of degradation fragments of different lengths. These ladders, which are subsequently separated in size and detected in the mass spectrometer, 30 provide higher sample throughput but the approach still requires a fair amount of up-stream wet chemistry. The need for this additional processing limits further improvement in the sensitivity and speed of sequence analysis.
  • an intact protein or polypeptide molecule is introduced into a mass spectrometer and allowed to accumulate in a linear ion trap.
  • Edman degradation reactions are then conducted in gas phase (within the linear ion trap) by introducing chemical reagents into the ion trap.
  • the cleavage products after each cycle are then ejected/extracted from the ion trap and their mass spectrum is determined.
  • the final conversion step used on condense-phase sequencing (where the ATZ derivative is converted in a PTH derivative; see Fig. 1) is unnecessary because there is no mass change involved in the conversion. This is the most ambitious of the three approaches, and is the focus of the present invention.
  • the invention thus combines the time-tested de novo sequencing capability of Edman degradation chemistry and the speed and sensitivity of mass spectrometry.
  • gas-phase coupling reactions and cleavage reactions analogous to the first two steps in condensed-phase Edman degradation were studied separately for small peptides using a modified triple quadrupole mass spectrometer.
  • Methylisothiocyanate (MITC) was used as the gas- phase Edman reagent.
  • Selective cleavage of the N-terminal peptide bond of the peptide derivative ion was achieved by collisional induced dissociation.
  • gas-phase Edman degradation is a promising approach for sequence analysis of intact protein/peptides.
  • the primary embodiment of the invention is thus a method of identifying an amino acid residue of a polypeptide or protein.
  • the method comprises performing a peptide degradation reaction on a polypeptide or protein ion reactant in the gas phase.
  • the reaction yields a first ion product corresponding to a first amino acid residue of the polypeptide or protein reactant, as well as a polypeptide or protein fragment ion.
  • the mass-to-charge ratio for the first ion product, or the polypeptide or protein fragment ion, or both is then determined.
  • the first amino acid residue of the polypeptide or protein reactant is identified from the mass-to-charge ratio so determined.
  • the method can be repeated reiteratively to determine part or all of an amino acid sequence of the polypeptide or protein reactant.
  • polypeptide or protein reactant is ionized via electrospray ionization.
  • electrospray ionization Other ionization methods can also be used, such as matrix-assisted laser desorption/ionization.
  • the peptide degradation reaction can be performed on a plurality of different polypeptide or protein reactants simultaneously.
  • the reaction yields a corresponding plurality of first ion products and a corresponding plurality of polypeptide or protein fragment ions.
  • the mass-to-charge ratios for the corresponding plurality of polypeptide or protein fragment ions is then determined.
  • the first amino acid residue of each polypeptide or protein reactant in the plurality can be determined from the mass-to-charge ratios so determined.
  • Another embodiment of the invention is directed to a method of determining an amino acid sequence of a polypeptide or protein.
  • the method comprises performing a gas-phase peptide degradation reaction on a polypeptide or protein reactant within an ion trap, wherein the reaction yields a first ion product corresponding to a first amino acid residue of the polypeptide or protein reactant.
  • the first ion product is then selectively transmitted from the ion trap into a mass spectrometer.
  • the mass-to-charge ratio for the first ion product is thus determined and the chemical identity of the first amino acid residue of the polypeptide or protein reactant is also determined (preferably by comparison to known standards).
  • the steps are then repeated to determine part or all of the amino acid sequence of the polypeptide or protein reactant.
  • the invention includes a method of determining an amino acid sequence of a polypeptide or protein, where the method comprises ionizing a polypeptide or protein reactant to yield a reactant ion and then trapping the reactant ion within a linear ion trap.
  • the polypeptide or protein reactant is then contacted with an Edman reagent to yield a thiocarbamoyl intermediate.
  • the intermediate is then subjected to collision-induced dissociation, or contacted with an acid, to yield a first ion product.
  • the first ion product is then selectively transmitted into a mass spectrometer to determine its mass-to-charge ratio.
  • a chemical identity for the first amino acid residue of the polypeptide or protein reactant is determined based on the mass spectrum so generated.
  • the invention is also directed to a mass spectrometer comprising: an ion trap comprising a quadrupole having selectively adjustable voltage and radio frequency, an entrance having selectively adjustable voltage, and an exit having selectively adjustable voltage, wherein the ion trap is dimensioned and configured to allow gas-phase reactions to take place therein.
  • the spectrometer includes a valve operationally connected to the ion trap, wherein the valve is dimensioned and configured to introduce reagents into the ion trap.
  • the device comprises a quadrupole mass analyzer operationally connected to the exit of the ion trap.
  • the linear ion trap comprises a quadrupole cell having a plurality of segments, wherein voltage and radio frequency within each segment is selectively adjustable independent of all other segments.
  • the linear ion trap further comprises an entrance having selectively adjustable voltage and an exit having selectively adjustable voltage.
  • the linear ion trap is dimensioned and configured to allow gas-phase reactions to take place therein.
  • a valve is provided to introduce reagents into the ion trap.
  • a charge reducer is operationally connected to the entrance of the ion trap.
  • a quadrupole mass analyzer is operationally connected to the exit of the ion trap, and an orthogonal time-of-flight mass analyzer is operationally connected to the quadrupole mass analyzer.
  • Fig. 1 is a reaction scheme depicting the sequential steps in conventional Edman degradation reaction.
  • Step I is the reaction of a phenylisothiocyanate (PITC) with the N-terminal residue of the protein to be sequenced.
  • Step II shows the formation of a phenylthiocarbamoyl (PTC) intermediate.
  • Step III illustrates the formation of the anilinothiazolinone (AZT) residue.
  • Steps IV and V illustrate the conversion of the AZT residue into a more stable phenylthiohydantoin (PTH) derivative.
  • Fig. 2 is a schematic drawing illustrating electrospray ionization (ESI).
  • ESI electrospray ionization
  • the very high electric field imposed by the power supply causes an enrichment of positive electrolyte ions at the meniscus of the solution at the metal capillary tip.
  • the net positive charge is pulled downfield by a negatively-charged electrode, thereby transforming the meniscus into a cone that emits a fine mist of positively-charged droplets.
  • Solvent evaporation reduces the volume of the droplets (which maintain their constant charge), which causes the droplets to fission.
  • Charge balance is attained by electrochemical oxidation at the positive electrode and reduction at the negative electrode.
  • Fig. 3 is a schematic diagram of a triple quadrupole mass spectrometer.
  • Ql and Q3 are mass-analyzing quadrupoles and q2 is a radio frequency-only (RF-only) quadrupole.
  • RF-only radio frequency-only
  • Fig. 4 is a typical a-q stability diagram.
  • the shaded area represent those areas in a-q space wherein correspond to stable solutions of Mathieu's differential equation.
  • Fig. 5 is a reaction scheme illustrating the generally accepted mechanism for the fragmentation of thiocarbamoyl derivatives of protonated peptides to yield modified b, and y n ., ions. 31
  • Fig. 6 is a schematic diagram of a mass spectrometer.
  • Fig. 7 is a schematic diagram of a cubic electrodynamic trap and related components.
  • Figs. 8 A, 8B, 8C, and 8D are graphs showing image charge (Q') or output voltage (Vout) as a function of time.
  • Figs. 8 A and 8C depict the image charge formed on the detection electrode of an inductive detector as a function of time.
  • Figs. 8B and 8D illustrate the output voltage generated upon detection of negative and positive ions, respectively.
  • Fig. 9 A is a mass spectrum of insulin (100 ⁇ M) acquired with a charge-sensing ring detector in positive ion mode;
  • Fig. 9B is a corresponding mass spectrum acquired with the ring detector in negative ion mode.
  • Fig. 10 is is a schematic diagram of a mass spectrometer according to the present invention.
  • Fig. 11 is a schematic diagram depicting the potential and timing parameters for a typical 35 ms trapping cycle.
  • Fig. 12 is a three-dimensional, cross-section through the axial yz plane of a segmented rod quadrupole ion trap.
  • Fig. 13 is a histogram depicting the ejection time distribution for m/z 200, 350, and 500 ions. The ejection time starts at the end of a 2 ms delay.
  • Fig. 14 is a graph depicting the timing sequence for gas-phase Edman degradation experiments within an ion trap as described herein.
  • the voltage values in parentheses are the preferred low voltage and high voltage toggle points on each element.
  • Figs. 15A, 15B, 15C, and 15D are mass spectra of gas-phase ion-molecule reactions of the Edman reagent MITC with singly- and doubly-protonated forms of the polypeptides MRFA and TLLELAR.
  • Fig. 15A singly-charged MRFA.
  • Fig. 15B doubly-charged MRFA.
  • Fig. 15C singly-charged TLLELAR.
  • Fig. 15D doubly- charged TLLELAR.
  • Fig. 16 depicts a reaction scheme for a possible mechanism for the gas-phase coupling reaction that involves an /nter-rmolecular proton transfer.
  • Fig. 17 depicts a reaction scheme for a possible mechanism for the gas-phase coupling reaction that involves an m/ra-molecular proton transfer.
  • Fig. 18 is a mass spectrum of the gas-phase ion-molecule reaction of MITC with the [M +H] + ion of isopropyl amine.
  • Fig. 19 depicts a reaction scheme for a possible mechanism involving an intermolecular proton transfer for the gas-phase coupling reaction between MITC and amino acids or peptides
  • Figs. 2OA and 2OB are mass spectra recorded following low-energy CID of the protonated tripeptide GLA (Fig. 20A) and the N-terminal MTC derivative of GLA (Fig. 20B).
  • Fig. 21 are mass spectra recorded following low-energy CID of the protonated tetrapeptide MRFA (Fig. 21 A) and the N-terminal MTC derivative of MRFA (Fig. 21B).
  • Figs. 22A, 22B, 22C, and 22D are mass spectra recorded following low-energy CID of the singly- and doubly-charged heptamer TLLELAR (Figs. 22A and 22B, respctively) and the N-terminal MTC derivative of singly- and doubly-charged TLLELAR (Figs. 22C and 22D, respectively).
  • the method disclosed provides a new technology for protein sequencing with high speed and sensitivity. While the washing and extraction steps in condensed-phase Edman degradation are lengthy and troublesome, manipulation of gas-phase ions is clean and fast. Moreover, the conversion step can be skipped in gas-phase degradation because it is the mass of the product ions that is is measured.
  • the sensitivity in conventional Edman degradation is mainly limited to the high femtomole level (300- 500 fm). Mass spectrometry is capable of detecting 10 s ions with microchannel plate detectors, which expands the sensitivity to the attomole level. Therefore, sequence information can be obtained for literally any protein spot detectable on a gel.
  • a “detector” is any device, without limitation, now known or developed in the future, that can detect ions. Explicitly included within the term “dectector” are channel electron multipliers (CEMs), microchannel plate detectors (MCPs) and inductive ion detectors.
  • CEMs channel electron multipliers
  • MCPs microchannel plate detectors
  • inductive ion detectors inductive ion detectors.
  • Edman reagent refers broadly to any compound capable of being used in the Edman degradation reaction to sequence polypeptides and proteins.
  • the term “Edman reagent” explicitly encompasses isothiocyanate-containing compounds, including, without limitation, substituted and unsubstituted alkyl-isothiocyanates ⁇ e.g. , methylisothiocyanate, ethylisothiocyanate, etc.), and susbstituted and unsubstituted aryl-isthiocyanates (e.g. , phenylisothiocyanate, halo-substituted phenylisothiocyanate, etc.). See Table 1. A host of different Edman reagents can be purchased from suppliers such as Sigma-Aldrich Chemicals, Milwaukee, WI.
  • ion refers to singly- or multiply-charged atoms, molecules, and fragments of molecules, or either positive or negative polarity.
  • ion also encompasses charged aggregates of one or more molecules or fragments or molecules.
  • ionization source ion source
  • ion source ionizer
  • MALDI matrix-assisted laser desorption/ionization
  • ESI electrospray ionization
  • Other types of ionizers include laser- induced ionization (in the condensed or liquid phases), corona discharge ionizers, and the like.
  • mass spectrometer or “mass analyzer” define any device used to determine the mass-to-charge ratio (m/z) of an ion in the gas phase. Examples include, but are not limited to, time-of-flight mass spectrometers, quadrupole mass spectrometers, and tandem and multi-stage mass spectrometers.
  • operationally connected when referring to two or more elements of a device indicates that the two elements communicate with one another (directly or indirectly, physically, electronically, electrically, or via a wireless connection, etc.) and function as defined with respect to one another. Elements that are “operationally connected” do not need to be physically or directly connected to one another.
  • selectively adjustable indicates an ability to select the value of a parameter over a range of possible values.
  • the value of a given selectively adjustable parameter can take any one of a continuum of values over a range of possible settings.
  • all machine settings referenced in the disclosure are selectively adjustable.
  • a schematic diagram of a first embodiment of a mass specrometer according to the present invention is shown schematically in Fig. 6.
  • the instrument shown in Fig. 6 utilizes time-of-flight mass analysis of large ions generated from individual charged droplets. Charged droplet production is accomplished with a piezoelectric droplet-on- demand dispenser. The charged droplet is held in an electrodynamic trap to desolvate.
  • a trapped droplet Once a trapped droplet reaches the desired state of desolvation, it (or the resultant gas- phase analyte ion) enters the high vacuum region of the mass spectrometer via an aerodynamic lens.
  • the aerodynamic lens sub-assembly functions to focus the analyte droplet or ion onto a central axis where an in-line TOF mass analysis is subsequently performed.
  • a series of inductive detectors are employed along the TOF axis to measure both the ion's initial velocity and the velocity after TOF acceleration to yield an accurate mass. The nondestructive nature of this new ion detection scheme allows further ion detection using more sensitive detectors or tandem MS to be performed.
  • Pulsed nanoelectrospray sources have been developed in the Lloyd Smith group at the University of Wisconsin-Madison. See U.S. Patent No. 6,906,322, issued June 14, 2005, to Berggren, Westphall & Smith, and U.S. Patent No. 6,797,945, issued September 28, 2004, to Berggren, Westphall, Scalf & Smith (the entire contents of which are incorporated herein).
  • the ion source is constructed from a glass capillary epoxied into a cylindrical piezoelectric element. A single droplet is released from the end of the capillary as a result of a rapid pressure pulsation generated by a radial contraction of the piezoelectric element.
  • the size of the droplet produced depends on the solution conditions, the orifice diameter, and the amplitude and duration of the pressure pulse which is controlled by the amplitude, duration and shape of an electronic pulse applied to the piezoelectric element.
  • the droplets are charged by inserting a platinum wire into the back end of the dispenser to hold the solution at high potential.
  • the ability to control the number and frequency of ionization pulses distinguishes this ionization technique from continuous ionization sources like ESI.
  • this technique has the advantages associated with the gentle nature of ESI, while avoiding the undesirable characteristics of the mutual charge repulsion leading to inefficiencies in sample introduction.
  • the droplets generated using the piezoelectric ionization source are often too large ( " 20 ⁇ m in diameter) to be completely desolvated before entering the high vacuum region of the mass spectrometer. Therefore a device is needed to extend the desolvation time.
  • An electrodynamic droplet levitation trap accomplishes this taks. The charged droplet is retained in the levitation trap until it has neared complete desolvation (/. e. , until the drop reaches the same size as ESI-generated droplets, generally 0.1 ⁇ m or less) at which point the droplet will exit the trap and be guided into the entrance port of the mass spectrometer by an aerodynamic lens.
  • An aerodynamic lens assembly comprising of a series of apertures with decreasing size, replaces the conventional nozzle-skimmer and collisional cooling regions used in typical ESI instruments.
  • Electrostatic lenses are often employed to collimate or focus an ion beam through apertures.
  • most lens systems exhibit aberrations of one type or another, such as minimizing the optimum focus conditions to a narrow m/z window over a limited energy range.
  • ions that are brought into focus through an aperture will quickly diverge on the far side of the aperture.
  • the aerodynamic lens stack 59"62 has the unique capability of being able to direct a droplet or particle, in the sub-micron size range, through a nozzle skimmer arrangement which exits into a region of high vacuum with the particle exiting on axis.
  • the lens stack utilizes the flow of a background gas (going from a region of higher pressure to a region of lower pressure) in place of electric potentials to transport and focus the ion.
  • the charged droplet is brought to the center axis due to its inertia, rather than its charge.
  • Inductive ion detectors have been developed for TOF measurement and can be used as detectors in the present invention. 15 ' 4 ⁇ 63"65 An inductive detector measures an image charge created on a conductive surface as the ion passes by the surface. The velocity of the ion can thus be determined from the timing information obtained as it passes through two inductive detectors with known separation. Electrodynamic droplet levitation has characteristics that interface nicely with the mass spectrometer disclosed herein; namely, the ability to direct the desolvated droplet, or its offspring daughter droplets, out of the trap along a defined axis. A cubic trap, illustrated schematically in Fig. 7, is preferred.
  • the opposing faces of a cube constitute three sets of planar electrodes, that is, each pair of opposing faces is an electrode set.
  • Each set of planar electrodes is driven by an AC voltage which is 120° out of phase with the other two.
  • two sets of planar electrodes may be driven 60° out of phase while the third set is held at ground.
  • a DC potential may be applied to a set of electrodes to generate a balance force between the plates.
  • Both plates in the electrode pair are driven with the same AC signal.
  • An aperture located in the center of the planar electrode allows access to the trap and is reported to have negligible effect on the electrodynamic characteristics of the trap for aperture diameters less than one eighth the length of the side of the planar electrode. (A circular aperture is preferred; apertures of other geometries can be utilized, provided they are suitable small so as not to disturb the electric fields generated by the electrodes.)
  • ⁇ represents any of the three axial displacement variables x, y and z.
  • E ⁇ is the AC component of the electric field
  • ⁇ is the viscosity of the medium in which the particle is immersed
  • q is the charge on the particle
  • r is the radius of the particle.
  • Equation 2 (8.3212/ a) (u/a - 0.5) Vac-cos ( ⁇ t) (2)
  • a the edge length
  • Vac the peak amplitude
  • ⁇ the frequency of the AC voltage
  • Equation 3 is a damped form of the Mathieu differential equation. This particular differential equation also describes the motion of an ion in a multipole field. Recall that the solution to this equation defines the stability region; see Fig. 4. Given a certain combination of ⁇ and Vac, only particles within the stability region (i.e., particles with a certain range of size, mass and charge) will be trapped. A droplet within the stability region, but initially not at the center of the trap will be brought into a stable oscillation in the central region of the trap and oscillate at the frequency of the AC signal applied to the electrodes.
  • a point of interest about the trap is that a droplet with enough energy to enter the trap will also have enough energy to exit the trap if the droplet were able to maintain its energy.
  • the viscous drag force due to air molecules within the trap removes energy from the droplet, thereby permitting it to obtain a stable trajectory inside the trap.
  • Fig. 7 shows a schematic diagram of a typical experimental setup.
  • a droplet dispenser is employed to introduce droplets into the trap. Charged droplets are formed by applying +1 kV to the solution electrode in the dispenser.
  • the droplet dispenser can dispense a single charged droplet or produce charged droplets continuously at low frequency ( " 1 Hz) as controlled by the frequency of the voltage pulses applied to the piezoelectric element.
  • the preferred cubic electrodynamic trap is constructed from six thin copper plates. Each plate has an edge length of 2.54 cm and a " center aperture that is 0.32 cm in diameter. Two non-conductive square frames are used to hold the six " copper plates in position and separate them from each other.
  • One pair of opposing plates are first embedded into frames, the other four plates, perpendicular to the two embedded plates, are then inserted into the four sides of each frame. Each pair of opposing plates are separated by 2.79 cm.
  • the pair of electrodes perpendicular to the vertical direction are referenced to ground, while the other two pairs are connected to AC voltages 60° out of phase.
  • the cubic trap drive electronics are briefly described as follows: Two sine waves are generated by a programmable analog voltage output device (National Instruments (Austin, Texax), Model PCI-6173). The sine wave frequency, amplitude, and phase shift are all software controllable. The output signals with maximum peak- to-peak amplitude of 20 V is fed into step-up transformers to produce the high voltage AC signals of several hundred to several thousand volts (peak-to-peak). The output signals from the transformers are AC coupled to DC high voltage converters which provide DC offset voltages to the electrode pairs. The high voltage DC offsets (0-200 V) can be regulated via analog signals (0-12 V) from the PCI-6713 board.
  • the size of a trapped droplet is measured from the image acquired using a CCD camera attached to a 6x microscope objective.
  • a single CCD pixel corresponds to an area of 16 ⁇ m 2 .
  • the droplet diameter can be measured.
  • the error associated with the measurement is ⁇ 4 ⁇ m based on the size of the individual pixels comprising an image.
  • the droplets are illuminated by an LED strobe driven at the same frequency as that of the electrodynamic trap (typically 60 Hz).
  • the initial size of a droplet as it is formed at the dispenser tip is measured in a similar manner by moving the CCD camera and the LED strobe from the central region of the trap to the dispenser tip.
  • the LED strobe is coupled to the droplet dispenser, that is droplets are generated continuously at the same frequency ( " 1 Hz) as that of the LED strobe.
  • a mechanical shutter is employed to block these droplets from entering the trap.
  • the shutter is removed, and the operational mode of the dispenser is switched from continuous droplet generation to single droplet generation.
  • Detection by charge induction can eliminate some of the problems associated with a MCP detector.
  • the inductive detector was positioned at the end of a TOF mass spectrometer and aligned with the ion beam axis. All spectra were obtained at an acceleration voltage of 25 kV.
  • a charge sensing grid detector was constructed which resembles that described by Park et al. 63
  • the detector comprised three 90% transmission grids (MN-17, InterNet, Inc. , New Hope, MN) applied to thin stainless steel rings using silver print (GC/Waldom Electronics, Inc., Rockford, IL).
  • the steel rings have an outer diameter of 6.99 cm and an inner diameter of 5.08 cm.
  • the grids are placed parallel to one another with the distance between adjacent grids being 0.57 cm.
  • the inner grid is the detection grid and the two outer grids are shielding grids.
  • the outer shielding grids are held at ground, while the center detection grid is connected to the gate of a 2N4416 FET.
  • Fig. 8A, 8B, 8C, and 8D The image charge, Q', and the output signal, Vout, generated upon detection of both positive and negative ions, are illustrated in Fig. 8A, 8B, 8C, and 8D as functions of time.
  • the transmission grids are removed from the charge sensing grid detector.
  • The converts the induction detector into charge-sensing ring detector since it comprises three ring electrodes as the shielding and detection elements.
  • Figs. 9A and 9B show representative MALDI-TOF spectra of insulin acquired in both positive-ion mode and negative-ion mode (respectively) with a charge-sensing ring detector. Comparing Figs. 9A and 9B with Figs. 8A-8D indicates that the response of the charge-sensing ring detector is indeed due to image charge formation. Note that Figs. 9A and 9B are mirror images of one another, clearly revealing the reversal of polarity between the two spectra.
  • This type of detector has an inherent peak broadening effect due to the separation between the shielding ring and the detection ring. For this reason, the distance between adjacent rings was decreased from 0.57 cm to 0.25 cm to improve the resolution.
  • Another geometry factor, the inner diameter of the rings has a direct impact on the total image charge induced. For an ion traveling on center axis, image charge formation is more efficient when smaller rings are used because the ion passes closer to the detection electrode. However, larger rings are more effective for ion collection (because the ion beam tends to diverge). It is found that a detector with rings of 2.54 cm i.d.
  • a linear ion trap was created by applying timed stopping potentials on the entrance and exit lenses of a RF-only quadrupole.
  • ions are trapped in the radial direction by the RF quadrupole field.
  • the axial direction trapping is accomplished by the blocking potentials at the entrance and exit lenses.
  • the z direction translational energy of an ion is determined by the energy dampening effects of collisions with the gas. A sufficient number of collisions will result in an ion whose translational energy in the z direction is determined by the thermal motion of the background gas.
  • the Q/LIT/Q mass spectrometer of the present invention is shown schematically in Fig 10.
  • the third quadrupole, q2 is 15.4 cm long and has a field radius of 6 mm.
  • Each rod in q2 is made out of several parallel wires which together form a cylindrical surface 8 mm in radius.
  • This construction of q2 allows the background buffer gas to traverse the quadrupole without obstructions, while confining ions inside the q2.
  • the initial kinetic energy of an ion as it enters q2 is determined by the DC offset difference between q ⁇ and q2 (5 eV for a singly charged ion at a typical setting). At these physical dimensions and settings, it takes 70 ⁇ s for a 5 eV ion of m/z 200 to travel through q2.
  • the voltages on the entrance and exit lenses are controlled by a circuit as illustrated in Fig. HB. In normal QqQ MS mode, the entrance and exit lenses are held at 10 V and 0 V, respectively.
  • the timing parameters of a trapping cycle are controlled by two pulse generators and is shown schematically in Fig. HA. Of the two pulse generators, one (pulse generator A in Fig.
  • HB provides the master clock and triggers a delayed pulse sequence from the second pulse generator (pulse generator B in Fig. HB).
  • the first phase of a trapping cycle is ion injection.
  • the potential on the exit lens is raised to a higher value (40 V) as controlled by pulse generator A.
  • the potential on the entrance lens is toggled to the same level as that on the exit lens (40 V) to prevent ions from both entering the trap and escaping from the trap in the z direction. This starts the trapping period, typically 10 ms to 1000 ms.
  • Ion ejection is achieved by lowering the voltage on the exit lens to 0 V, while keeping the voltage on the entrance lens high (40 V) for a period of 10 ms to 100 ms.
  • An important feature of this design for the driving electronics is its flexibility in different operation modes. The Q/LIT/Q mode can be switched to the normal QqQ mode simply by turning off the pulse generators.
  • Ions are trapped in the radial direction by the RF quadrupole field.
  • the axial direction trapping is accomplished by applying blocking potentials at the entrance and exit aperture.
  • protein ions injected into ion trap lose their translational energy in the axial direction upon collisions with a neutral target and thus are trapped inside the potential well.
  • the trapping process can be simulated using SIMION 3D-brand software (version 7.0), distributed by Scientific Instrument Services, Ringoes, NJ.
  • the RF potential applied to the quadrupole rods is defined by:
  • V V ri cos(2 ⁇ ft) (4)
  • V rf is the RF amplitude
  • f is the RF frequency in Hz
  • t is the flight time of the ion.
  • the rods are coupled so there is a phase difference of ⁇ between the values of V rf applied to the two pairs of opposite electrodes.
  • One important feature is the use of a 3D hard sphere collision model for simulating the ion-buffer gas interactions.
  • the collision frequency for an ion is calculated using the following equation:
  • V re is the averaged relative velocity between the ion and the buffer gas
  • N is the gas number density
  • the buffer gas molecules may move toward the ion or leave the ion along the direction of the relative velocity before they collide, statistically the ion and buffer gas approach the collision point at a 90° angle relative to each other:
  • V rel (V? + F 2 2 ) (6)
  • Vj and V 2 are the average velocities of the ion and buffer gas molecule between collisions.
  • Gas number density is calculated from its pressure, which can be adjusted at the beginning of each run.
  • the collision cross section of incident ions is assumed to be IOOOA 2 (singly-charged ubiquitin ion), 35 ' 36 and the radius and molecular weight of the buffer gas molecule is assumed to be 2 A and 40 a.u. (argon).
  • the lab frame velocity of buffer gas, V 2 is defined by:
  • V 1 is calculated by dividing the path the ion has passed since the last collision (L p ) by the time of this period (T p ). V 1 is then used to calculate Z and ⁇ . If L 1 , is larger than ⁇ , then there is a collision. If not, the current time step ( ⁇ t) is added to T p and ⁇ t*V, is added to L 1 ,. V 1 is the current velocity of the ion. This process repeats until L 1 , is larger than ⁇ and a collision occurs. T p and L p are then set to 0 and the above steps are repeated to determine the next collision event.
  • the new magnitude and direction of ion velocity is calculated and assigned to the ion.
  • E int is the energy transferred to internal energy of the collision partners
  • ⁇ cm is the scattering angle in the center-of-mass coordinates.
  • E int is approximately given by the center of mass energy E CM .
  • Eqn. (10) is used to calculate the magnitude of ion velocity after collision, which is given by:
  • the velocity of the center of mass in lab frame is given by:
  • the angle of ⁇ defines a cone of scattering surrounding the original velocity vector. A random point on the cone is generated to define the new azimuth and elevation angle of the post-collision ion trajectory.
  • DC voltage on the entrance and exit apertures is 5 V and 10 V respectively.
  • the argon gas pressure can be altered to investigate trapping efficiencies at different gas density. With a sufficient number of collisions, an ion will loss part of its initial kinetic energy and be trapped inside the potential well. Damping an ion's translational energy continues until the ion reaches its thermal equilibrium. In a quadrupole field, a trapped ion oscillates at specific frequencies determined by its m/z ratio: 37
  • n is an integer
  • ⁇ is given approximately by:
  • Resonant excitation can be used either to remove unwanted ions, or to increase ion kinetic energy to promote ion-molecule reaction or collision-induced dissociation.
  • the collision frequency is increased due to the increased ion velocity and a larger amount of internal energy is stored after each collision which will be available for later reactions.
  • the ion oscillation amplitude exceeds r 0 , the ion will escape from the trap or collide with one of the poles. While pulsed or off-resonance irradiation is used to facilitate reactions, a continuous resonant irradiation is used to eliminate unwanted ions, e.g., protein ions at higher charge states.
  • Resonant excitation was modeled using the SIMION 3D-brand software. Actual resonant frequency is found to be 3% higher than the calculated value using Eqn. (21).
  • the motion of a m/z 5000 ion upon resonant excitation shows the amplitude of excitation AC waveform is 1 V, with a frequency 6.37 kHz. If the excitation source frequency is shifted even slightly, the ion will oscillate with a periodically changing amplitude.
  • an ion having a m/z of 5000, excited at 6.31 kHz (1 % lower than its resonant frequency) has an average energy when passing the center xz plane of 0.3 eV, about one half of D, and 10 times higher than its thermal energy.
  • This off- resonance excitation provides a simpler operation because a continuous irradiation can be used.
  • the molecular weight of the protein of interest is measured to determine the excitation frequency in the first degradation cycle.
  • the excitation frequency is then adjusted (increased) accordingly as the mass of the parent ion decreases after each cycle of degradation.
  • Peptide fragmentation can be suppressed by fixing the site of the charge within the ionized peptide. Because a singly-charged ion is most likely to have the proton localized to the most basic site on the ion, one approach is to conduct Edman degradation on singly-charged protein ions only. However, large protein ions usually have a very low abundance at the singly charged state. For this reason, charge reduction techniques 32"34 are used to reduce the charge state of protein ions.
  • a charge reduction chamber/reaction chamber 14 (see left-hand side of Fig. 10) is positioned between the ionization source 12 and the entrance of the triple quadrupole mass spectrometer 100.
  • gas molecules N 2 or CO 2
  • ionized gas molecules normally in a corona discharge or by exposure to a 210 Po ⁇ -particle source. Protein ions entering the chamber are neutralized by these ionized gas molecules. Consequently, ions leaving the charge reduction chamber have higher abundance of ions low charge states.
  • Singly-charged protein ions usually have a rather high m/z ratio, and band-pass filtering at such a high mass range is impractical for quadrupole mass filters.
  • Ql (20) is operated in an RF-only mode and all of the ions with m/z ratios higher than the low-mass cut-off value (LMCO) will transmit and enter the collision cell 22 ⁇ i.e., the linear ion trap).
  • LMCO low-mass cut-off value
  • Each gas-phase Edman degradation cycle includes three major steps: (i) a coupling reaction of protein ions with PITC, (ii) a cleavage reaction of FTC derivatives, and (iii) a mass measurement of AZT derivatives. See Fig. 1. Because conversion of AZT to PTH is slow compared to ion detection, and because there is no mass difference between AZT and PTH derivatives, conversion is not necessary in gas- phase degradation.
  • the argon buffer gas pressure in Q2 and Q3 is maintained at 2 mTorr.
  • ions are preferably generated by pneumatically assisted electrospray (+4 kV), although any type of ion generator 12 now known (ESI, MALDI, electron discharge, etc.) or developed in the future may be utilized.
  • the ions pass through the charge reduction chamber 14 at +250 V. Ions at lower charge states are sampled at a nozzle/ skimmer assembly 16 (+ 15 V) that leads to the first low pressure region of the mass spectrometer (1 to 10 Torr).
  • a typical broadband waveform will span frequencies from 1 kHz to 300 kHz (30 kDa to 100 Da), created by a comb of sine waves each with an amplitude of 10 V and separated by a frequency of 500 Hz.
  • a typical notch in the broadband waveform is 1 kHz wide and is centered on the resonant frequency corresponding to the ion of interest.
  • Linear ion traps of different design to the one described here have been used for ion storage. 38"40 However, some important characteristics of ion traps remain unexplored.
  • charge capacity for a quadrupole 10 cm long is estimated to be ⁇ 10 10 electrons.
  • Experiments designed to investigate the trapping efficiency and charge capacity of this linear ion trap measure the ion current entering the trap using a charge detector (a tube 41 or simply a metal plate) placed at the end of the spectrometer. All the quadrupoles are operated at high pass mode.
  • gas-phase phenyl isothiocyanate (PITC) is introduced into the ion trap at pressure of 1-10 mTorr through a computer controlled valve (26 in Fig. 10). To facilitate the reaction, ions are excited by off-resonance irradiation. At the end of the coupling step, the PITC gas is pumped away in approximately 2-3 seconds.
  • the coupling step is studied individually by injecting protein or peptide ions into the PITC gas-filled trap. After a certain reaction period, all the ions are ejected for mass measurement to obtain the relative abundance of reactant and products.
  • Penta-fluoro- 508.5 c 12.8 a. CRC handbook of chemistry and physics, 70th Edition b. Acros Organics, Catalog of organics and fine chemicals, Fisher Scientific, 2002 c. Ref. M d. Lancaster Catalog, 2000 e. Estimated using group contribution values from Ref. 36 f. Unless otherwise notified, vapor pressures were calculated using an empirical formula: ln[p(atm)] l g. NIST chemistry webbook
  • TFA is a strong volatile acid which has been used successfully in gas-phase sequencers.
  • the TFA gas pressure is maintained at 1 to 10 mTorr, and off-resonance excitation is applied.
  • PTC derivatives are prepared in solution first, then studies similar to that on coupling reaction can be conducted.
  • the buffer gas pressure is adjusted without introducing TFA. Cleavage is achieved by collision-induced dissociation. PTC derivatives have a specific fragmentation pattern that breaks only the N-terminus peptide bond. 44"46
  • the proposed mechanism for fragmentation of PTC derivatives is similar to that of a protein ion (see Fig. 5). Because the proton is localized after charge reduction, the energy requirement for cleavage of the N-terminal peptide bond in PTC derivatives is also enhanced. This can be accounted for by using a longer reaction time (ions are trapped instead of passing through) and excitation. Given the fact that modified b, ions (and its counter part y n . t ions) were formed almost exclusively when a mobile proton is available, it is possible to allow dissociation of PTC derivatives while minimizing nonspecific fragmentation of protein ions.
  • the minimum number density of MeOH 2 + required is 10 10 m '3 .
  • the total number of MeOH 2 + required is 10 5 .
  • the charge capacity of a linear ion trap of the stated volume is approximately 10 10 e.
  • the proton will be transferred to the most basic residue of the neutral fragment, either directly from MeOH 2 + or through an intramolecular bridge. 49 Ion-ion repulsion prevents any additional protonation to the already charged peptide. After reionization, singly-charged ions are formed with their proton localized at some basic site.
  • one DC supply is used to provide an offset to segment 1
  • a second floating DC supply connected across a voltage divider provides a constant electric field along the axis.
  • Fig. 12 is a cross-section view through the axial yz plane of the segmented rod.
  • a DC voltage of 5 V is applied to the entrance aperture and the exit is at ground.
  • the offset between segments is set at 0 V.
  • a stepped field is formed inside the ion trap by applying a proper DC offset that steadily decreases going from the entrance to the exit of the trap (thereby accelerating the ions out of the trap).
  • the buffer gas pressure is maintained at 2 mTorr
  • the DC voltage at the entrance aperture is maintained at 10 V
  • the DC offset at each segment is set at 8 V
  • the voltage on the exit aperture is maintained at 10 V for 2ms before an AC potential of 5 V (20 kHz) is applied and the DC offset is lowered to 2 V.
  • m/z 200 ions 100 A 2
  • m/z 350 ions 175 A 2
  • m/z 500 ions 250 A 2
  • Fig. 13 The simulated distribution of ejection times for m/z 200 ions (100 A 2 ), m/z 350 ions (175 A 2 ), and m/z 500 ions (250 A 2 ) are shown graphically in Fig. 13).
  • 100 ions were located evenly along the quadrupole axis with no initial kinetic energy.
  • Similar simulation on m/z 1000 ions (400 A 2 ) showed that there is no ejection up to 8 ms.
  • the excess MeOH 2 + ions are completely ejected and eliminated in Q3 (see Fig. 10), where LMCO is 100.
  • Q3 can be scanned at a certain rate ( " 5500 u/s) in a mass-to-charge range of from about 100 to about 350.
  • a portion of the product ion passes through Q3 and is detected by an ion detector, such as a microchannel plate detector (MCP, reference number 36 in Fig. 10).
  • MCP microchannel plate detector
  • a drawback of this detection scheme is the loss of sensitivity because the MCP only registers a small portion (in this case only 1/250) of product ions.
  • MCP microchannel plate detector
  • Another problem is that a quadrupole mass filter requires a continuous ion source. Ions ejected from a linear ion trap do not form a uniform and continuous stream. This leads to mass-dependent detection efficiency.
  • improved sensitivity can be achieved by coupling an orthogonal acceleration time-of-flight mass spectrometer 200 to the triple quadrupole system, as shown in Fig. 10. 49
  • the TOF chamber 200 is coupled to the quadrupole Q3 via an aperture ("A" in Fig. 10) and focusing lenses 32.
  • the aperture serves as the exit of the Q3 quadrupole and the differential pumping aperture between the quadrupole and TOF chambers.
  • the TOF source region has the same DC offset as Q3, so the axial ion energy in the TOF source remains small. It is preferable to operate Q3 at relatively high pressure. The benefit from the resultant collisional cooling effect is three-fold. First, it damps the translational energy of the ions, thereby creating a slower ion beam. This leads to higher duty cycle because duty cycle is defined as the ratio of the source filling time to the time between the acceleration pulses.
  • a slower ion beam gives a higher ion density to each pulse accelerated into the flight tube, thus enhancing sensitivity.
  • collision cooling creates a highly-collimated ion beam with small spatial and energy spread in the radial direction, which improves resolution in the TOF MS.
  • product ions ejected from the ion trap are focused on the axis in Q3 and enter the source region of the TOF chamber.
  • High voltage pulses (+5 kV, 30-100 ⁇ s) are applied to an acceleration grid 34.
  • the pulses are longer in duration than the flight time of a m/z 500 ion.
  • the signal from MCP is digitized, and the peak value used to calculate the mass of residue y n . t ion. This mass is subsequently used to calculate the oscillating frequency applied in the off-resonance excitation of the next cycle. These calculations are done automatically after each cycle and sent to a programmable AC waveform generator.
  • Post-Ionization, Pre-Mass Analysis, Gas-Phase Reaction In another approach, rather than performing the degradation reaction within an ion trap, the degradation reaction takes place immediately after the ionization of the protein or polypeptide reactant, and immediately prior to introducing the product ions into a mass analyzer. Thus, for example, immediately after ionization at the spray tip 12 (see Fig. 10), the ions pass into reaction chamber 14. The degradation reaction then takes place within chamber 14, and the product ions are passed into the mass analyzer 100, where the m/z of the first ion product, or the polypeptide or protein fragment ion, or both, is determined. In this approach, q2 in Fig. 10 is operated as a regular quadrupole (either as a mass-scanning quadrupole or in rf-only mode), rather than as an ion trap. Timing Sequence:
  • FIG. 14 A general timing sequence for degradation experiments is shown in Fig. 14.
  • Q2 and Q3 are operated in RF-only mode with V rf of 1 kV and an RF frequency of 3.1 MHz and 1 MHz respectively.
  • QO is also operated in RF- only mode, but with V rf of 100 V and an RF frequency of 1 MHz to transmit MeOH 2 + .
  • Ql is switched between two operation modes: a band pass at m/z 33 during pumping interval 1 and a high pass at all other times.
  • the frequency of the tickling voltage (for off-resonance excitation) is adjusted between cycles and between the coupling and cleavage steps in each cycle.
  • TLLELAR polypeptide SEQ. ID. NO: 1
  • AU reagents, amino acids and peptides, except the TLLELAR polypeptide (SEQ. ID. NO: 1) were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification.
  • TLLELAR SEQ. ID. NO: 1 was synthesized by the Peptide Synthesis Facility in the Biotechnology Center at the University of Wisconsin-Madison.
  • the MTC derivatives of peptides were prepared by mixing 200 ⁇ l peptide solution (5 mM in water), 400 ⁇ l pyridine, 1 ⁇ l triethylamine, and 10 ⁇ l methylisothiocyanate (melted by warming at 5O 0 C) in a 1.5 ml polypropylene tube. 51 - 52 The sample was incubated at 50 0 C for 30 minutes, then dried in a rotary evaporator. To remove any remaining traces of volatile substances, 1 ml ethyl acetate was added to the tube. After vortexing for 10 seconds, the ethyl acetate was removed in the rotary evaporator, leaving the dry coupled peptide in the tube. The product was dissolved in 1 : 1 water/acetonitrile (500 ⁇ l) containing 1 % acetic acid.
  • the work presented here was performed on an extensively modified commercial triple quadrupole mass spectrometer (API III, Perkin-Elmer Sciex Instruments, Wellesley, MA).
  • the quadrupole collision cell (q2) was modified into a Linear Ion Trap (LIT) as described earlier.
  • a relatively large voltage (170 V) between the orifice and QO was used to reduce solvent adduction.
  • a typical collisional energy is 5 eV for singly- charged ions, as determined by the voltage difference between q ⁇ and q2.
  • the timing parameters used in trapping cycles were: 10 ms for ion injection, 10 ms for ion trapping, and 10 ms for ion ejection.
  • MITC was used as the gas-phase Edman reagent and argon gas was used as the collision gas in the study of gas-phase cleavage reactions.
  • a three-way valve was added to the background gas inlet line, which allowed introduction of MITC and argon gas alternatively.
  • the MITC reagent was kept in its liquid form by maintaining the container at 50 0 C.
  • the instrument had a positive pressure ESI source, which comprises a long fused-silica polyimide-coated capillary (150 ⁇ m od; 25 ⁇ m id).
  • the inlet of the capillary was immersed in analyte solution contained in a 0.5 ml polypropylene tube.
  • a positive pressure of 10 p.s.i. was applied to the sample container.
  • the solution was maintained at a potential of +4,500 V for positive ion mode and -4,500 V for negative ion mode through a platinum electrode immersed in the sample.
  • the spray was stabilized with a sheath gas of N 2 (0.6 L/min) fed through a stainless steel tube (1.5 mm id) concentric with the silica capillary.
  • the spray end of the capillary was positioned 1.5 cm away from the ion-sampling nozzle of the mass spectrometer.
  • Amino acid and peptide ions generated by ESI were mass selected by Ql and allowed to react with MITC gas in the LIT.
  • the mass-to-charge ratios (m/z) of the product ions were measured by scanning the Q3 as ions were ejected from the trap.
  • Amino Acids PA a Relative abundance (%) and Peptides (kcal/mol) [M +H] + [M +HH-MITC] +
  • mechanism I a proton is transferred from the peptide ion to MITC, which initiates the nucleophilic attack of the N-terminal amino group to the thiocarbonyl group of MITC, yielding the coupling product ion.
  • mechanism II the amide oxygen is protonated, followed by nucleophilic attack of the N- terminal amino group to the thiocarbonyl group of MITC.
  • the first step in both mechanisms involves mobilization of a proton from a basic site, such as the side chain of a basic residue or the N-terminal amino group, to a less basic site, such as the nitrogen atom in MITC or the amide oxygen. Both proposed mechanisms explains why the coupling reaction is more efficient when a "mobile proton" is present.
  • a gas-phase reaction of MITC with isopropyl amine was performed.
  • the resultant mass spectrum is shown in Fig. 18. Because isopropyl amine does not have a carbonyl group, the coupling product ion observed could only have been formed via mechanism I (see Fig. 16). Further, the protonated MITC ion came from an intermolecular proton transfer from the peptide ion to MITC.
  • the proton affinity of isopropylamine 49 is 216-219 kcal mol "1 , which lies between those of G and G 2 .
  • the initial step in the coupling reaction of amino acids or peptides with MITC may be intermolecular proton transfer (mechanism III, shown in Fig. 19) or intramolecular proton transfer (mechanism II, shown in Fig. 17).
  • the low-energy collision product ion spectra of [M +H] + ion of the peptide MRFA and its MTC derivative under identical collision conditions are shown in Figs. 21A and 21B, respectively. These two spectra show little fragmentation, and, more importantly, no promotion effect on modified b ⁇ ion formation was observed for the MTC derivative (see Fig. 21B). This is attributable to the preferential location of the ionizing proton on the arginine side-chain.
  • the product ions observed (albeit in low abundance) include modified b,, y 2 and y 3 . Further, the high-mass fragments can be attributed to loss of CH 3 NH 2 from the derivative group of the precursor ion.
  • Figs. 22A, 22B, 22C, and 22D are the spectra for the corresponding N-terminal MTC derivatives of singly- and doubly-charged TLLELAR.
  • the CID production spectra for both singly- and doubly-charged ions of the native peptide was compared to the MTC derivative of TLLELAR (again, see Figs. 22A-22D). Similar to the other arginine-containing peptide, MRFA, the singly-protonated ions, underivatized (Fig. 22A) and MTC-derivatized (see Fig. 22C), show low fragmentation efficiencies due to the localization of the ionizing proton on the side chain of the arginine residue. In the case of the doubly-protonated underivatized peptide (see Fig. 22B), extensive fragmentation was observed.
  • the present invention therefore demonstrates that gas-phase reactions between Edman degradation reagents and protonated peptide ions yields Edman-type derivatives in a fashion directly analogous to the first coupling step in a condensed-phase Edman degradation.
  • the proposed mechanism for the formation of MTC derivative involves proton transfer. Consequently, the coupling reaction with MITC is more efficient for multiply protonated peptides.
  • the present invention further demonstrates that conversion of peptides to the N- terminal Edman-type derivative introduces a marked propensity for the formation of the derivatized b,, and complementary y ions, to the extent that other fragmentation pathways are not observed under the conditions of low energy collisional activation.
  • the mechanism for formation of modified b, ion from [M+nH] n+ ion of the peptide MITC derivative closely resembles that proposed for peptide fragmentation, in which the "mobile proton" plays a key role.

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Abstract

L’invention concerne un procédé et un dispositif rapides et sensibles pour séquençage de protéine. Le procédé utilise une combinaison de chimie de dégradation Edman et de spectrométrie de masse pour séquencer les protéines et polypeptides. Une réaction de dégradation de peptide est réalisée sur un produit réactif ionique polypeptide ou protéine en phase gazeuse. La réaction produit un premier produit ionique correspondant à un premier résidu d’acide aminé du produit réactif polypeptide ou protéine et un ion de fragment polypeptide ou protéine. Le rapport masse/charge pour le premier produit ionique ou l’ion de fragment polypeptide ou protéine, ou les deux, est ensuite déterminé. Le premier résidu d’acide aminé du produit réactif polypeptide ou protéine est alors identifié à partir du rapport masse/charge ainsi déterminé.
PCT/US2005/025185 2004-07-16 2005-07-15 Séquençage de protéine/peptide par dégradation chimique en phase gazeuse Ceased WO2007015690A1 (fr)

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US20220120758A1 (en) * 2019-06-06 2022-04-21 Fudan University Method and device for protein sequence analysis
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009073505A3 (fr) * 2007-11-30 2009-09-03 Wisconsin Alumni Research Foundation Procédés de traitement de données de spectres de masse en tandem pour une analyse de séquence d'une protéine
US8278115B2 (en) 2007-11-30 2012-10-02 Wisconsin Alumni Research Foundation Methods for processing tandem mass spectral data for protein sequence analysis

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